Methods of treating infertility

ABSTRACT

The present invention relates to improved assisted reproductive technology using highly purified menotropin (HP-hMG) to stimulate follicle development in controlled ovarian stimulation, particularly in women at risk of a high ovarian response to controlled ovarian stimulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/555,688, filed Aug. 29, 2019 now U.S. Pat. No. 11,351,228), which isa continuation of U.S. application Ser. No. 15/053,116, filed Feb. 25,2016 (now U.S. Pat. No. 10,413,592), which claims priority to U.S.Provisional Patent Application No. 62/121,113, filed Feb. 26, 2015; U.S.Provisional Patent Application No. 62/121,131 filed Feb. 26, 2015;European Patent Application No. 15157661, filed Mar. 4, 2015; andEuropean Patent Application 15185191, filed Sep. 15, 2015, the contentsof each of which are incorporated herein by reference in theirentireties.

FIELD

The methods described herein relate to assisted reproductive technology.In particular, described herein are methods for treating infertility,including controlled ovarian stimulation methods that may beparticularly useful for women at risk of a high ovarian responsethereto.

BACKGROUND

Assisted reproductive technology (ART) procedures generally involvestimulating egg production, harvesting eggs from a woman's ovaries,combining them with sperm in vitro, and transferring them to a woman'suterus (the donor or another woman). Success of ART is hampered bymaternal and perinatal risks associated with the stimulation of eggproduction, such as ovarian hyperstimulation syndrome (OHSS) and ectopicpregnancy. Other concerns that arise in ART are the production ofquality embryos and euploid blastocysts to support ongoing pregnancyrates and live birth rates.

Gonadotropins, such as menotropin (e.g., human menopausal gonadotropin,or hMG), follicle-stimulating hormone (FSH) and luteinizing hormone(LH), have been widely used for controlled ovarian stimulation (COS),and highly purified menotropin (HP-hMG) and recombinant human FSH(r-hFSH) have been used more recently. The efficacy of ovarianstimulation protocols may be enhanced using long gonadotropin hormonereleasing hormone (GnRH) agonists or GnRH antagonists for cycle control.See, e.g., Devroey et al. Fertility and Sterility 97: 561-71 (2012).

Because patient responses to ovarian stimulation vary widely, treatmentsoften are individualized. For example, individualization may be based onpredicted ovarian response to gonadotropin stimulation, which forecastspoor, normal or high response. High ovarian responders usually aredefined as women who produce high numbers of developing folliclesfollowing a standard protocol of controlled ovarian stimulation (COS).Although these patients are generally considered good candidates forART, high ovarian response may be associated with lower implantationrates and higher miscarriage rates, and thus a decreased chance ofsuccessful outcome as compared with a normal ovarian response. Thesehigh responders also are at greater risk for OHSS and the complicationsassociated therewith.

It has been hypothesized that ovarian stimulation may have a generalnegative impact on embryo quality, as assessed by morphologicalparameters and/or chromosomal analysis (e.g., euploidy vs. aneuploidy).See, e.g., Hamdine et al., “Ovarian Stimulation for IVF: MildApproaches” in Human Fertility: Methods and Protocols, Methods inMolecular Biology, Vol. 1154, Springer Science+Business Media, New York(2014). Additionally, Haaf et al., Fertility and Sterility 91(3): 733-38(2009), reported that a high oocyte yield is associated with anincreased chromosome error rate. However, Fatemi et al., HumanReproduction 28(2) 442-52 (2013), reported that high ovarian responsedoes not jeopardize ongoing pregnancy rates, but rather increasescumulative pregnancy rates.

Efforts to develop improved ART methods have involved exploring milderstimulation protocols. For example, Rubio et al., Human Reproduction25(9): 2290-2010 (2010), reported that decreasing the gonadotrophin doseadministered to high responders could improve fertilization rates andembryo quality, although the lower doses resulted in a decreased numberof oocytes. Other efforts have considered whether the specificgonadotrophin used impacts the results. For example, Ziebe et al., HumanReproduction 22(9) 2404-13 (2007), reported that the use of HP-hMGversus rFSH could impact the morphology of embryos, and observedimproved implantation, ongoing pregnancy and live birth rates among thetop-quality embryos derived from stimulation with HP-hMG compared withrFSH. However, Ziebe's findings were based on a visual assessment ofoocyte/embryo morphology on day three following retrieval, not based onchromosomal analysis, and morphology at day three is not correlated withaneuploidy. Along the same lines, La Marca et al., Fertility andSterility O-169 (2012), reported that among predicted high responders(subjects having an AMH≥5.2 ng/ml) the group stimulated with rFSH grouphad significantly more oocytes retrieved, but a significantly lower livebirth rate per cycle as compared to the group stimulated with HP-hMG.

There remains a need, therefore, for improved assisted reproductivetechnology methods, particularly for women at risk of a high response tocontrolled ovarian stimulation.

SUMMARY

In accordance with some embodiments, there are provided assistedreproductive technology methods useful for women at risk of a highresponse to controlled ovarian stimulation, comprising selecting a womanat risk of a high ovarian response to controlled ovarian stimulationhaving a serum anti-Müllerian hormone (AMH) level greater than or equalto 5.0±0.5 ng/ml, when measured using a Beckmann-Coulter Gen 2 assay ora comparable AMH level measured by a different method, and administeringto the selected woman an amount of highly purified menotropin (HP-hMG)effective to stimulate follicle development. In accordance with someembodiments, the methods are for improving the quality of an embryoproduced by in vitro fertilization after controlled ovarian stimulationin a woman at risk of a high ovarian response thereto. In accordancewith other embodiments, the methods are for increasing the proportion ofeuploid blastocysts produced by in vitro fertilization after controlledovarian stimulation in a woman at risk of a high ovarian responsethereto. In accordance with other embodiments, the methods are fortreating infertility in a woman at risk of a high ovarian response tocontrolled ovarian stimulation. In accordance with other embodiments,the methods are for effecting controlled ovarian stimulation in a womanat risk of a high ovarian response thereto. In accordance with otherembodiments, the methods are for increasing ongoing pregnancy rates. Inaccordance with other embodiments, the methods are for increasing livebirth rates. In accordance with other embodiments, the methods are forpromoting the development of euploid blastocysts, such as for futureblastocyst transfer, in a woman at risk of a high ovarian response tocontrolled ovarian stimulation.

In accordance with other embodiments, there are provided assistedreproductive technology methods useful for women at risk of a highresponse to controlled ovarian stimulation, comprising administering anamount of highly purified menotropin (HP-hMG) effective to stimulatefollicle development to a woman selected as being at risk of a highovarian response to controlled ovarian stimulation, wherein the selectedwoman has a serum anti-Müllerian hormone (AMH) level greater than orequal to 5.0±0.5 ng/ml, when measured using a Beckmann-Coulter Gen 2assay or a comparable AMH level measured by a different method. Inaccordance with some embodiments, the methods are for improving thequality of an embryo produced by in vitro fertilization after controlledovarian stimulation in a woman at risk of a high ovarian responsethereto. In accordance with other embodiments, the methods are forincreasing the proportion of euploid blastocysts produced by in vitrofertilization. In accordance with other embodiments, the methods are fortreating infertility in a woman at risk of a high ovarian response tocontrolled ovarian stimulation. In accordance with other embodiments,the methods are for effecting controlled ovarian stimulation in a womanat risk of a high ovarian response thereto. In accordance with otherembodiments, the methods are for increasing ongoing pregnancy rates. Inaccordance with other embodiments, the methods are for increasing livebirth rates. In accordance with other embodiments, the methods are forpromoting the development of euploid blastocysts, such as for futureblastocyst transfer, in a woman at risk of a high ovarian response tocontrolled ovarian stimulation.

In specific embodiments of any of these methods, the woman may have anantral follicle count (AFC) of greater than or equal to 10, or greaterthan or equal to 15, in both ovaries combined, prior to stimulation.

In specific embodiments of any of these methods, the method may resultin an increased proportion of euploid blastocysts, and/or an increasedproportion/percentage of euploid blastocysts versus aneuploidblastocysts, and/or a reduced aneuploidy rate, and/or an increasedongoing pregnancy rate, and/or an increased live birth rate, each ascompared to a comparable method using recombinant follicle-stimulatinghormone (rFSH) as the gonadotropin. In further specific embodiments ofany of these methods, the method may result in an increased proportionof euploid blastocysts, and/or an increased proportion/percentage ofeuploid blastocysts versus aneuploid blastocysts, and/or a reducedaneuploidy rate, and/or an increased ongoing pregnancy rate, and/or anincreased live birth rate, each as compared to a comparable method usingrecombinant follicle-stimulating hormone (rFSH) as the gonadotropin.

In specific embodiments of any of these methods, the amount of HP-hMGadministered may be from 100-300 IU per day for from about 1 to about 20days, or from 100-200 IU per day for from about 1 to about 20 days.

In specific embodiments of any of these methods, the HP-hMG may beadministered for at least 5 days.

In specific embodiments of any of these methods, the methods may furthercomprise administering a gonadotropin hormone releasing hormone (GnRH)agonist or a GnRH antagonist.

In specific embodiments of any of these methods, the methods may furthercomprise one or more of administering an amount of hCG effective totrigger ovulation, harvesting oocytes from the woman, and in vitrofertilization of harvested oocytes (optionally, by intra-cytoplasmicsperm injection (ICSI)).

In specific embodiments of any of these methods, the methods may furthercomprise assessing the chromosomal quality of blastocysts obtained fromthe woman after in vitro fertilization of oocytes harvested from thewoman. In further specific embodiments, the methods may further comprisetransfer of a blastocyst determined to be a euploid blastocyst. Inspecific embodiments, the transfer may be fresh transfer. In furtherspecific embodiments, the methods may further comprise freezing ablastocyst determined to be a euploid blastocyst.

The foregoing general description and the detailed description areexemplary and explanatory and are intended to provide furtherexplanation of the invention. For detailed understanding of theinvention, reference is made to the following detailed description ofspecific embodiments, taken in conjunction with the accompanyingfigures. Other objects, advantages and novel features will be readilyapparent to those skilled in the art from the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) the occurrence of high ovarian response (≥15 oocytesretrieved) and (B) live birth rates (LBR) among women classified aspotential high responders by a high initial anti-Müllerian hormone (AMH)level. Values within bars are n/total. p Values are based on theChi-Square Test. The percentage of women with a high ovarian response issignificantly lower for HP-hMG compared with rFSH in both the longagonist and the antagonist protocols.

FIG. 2 sets forth a flow chart of the study design for the clinicaltrial of Example 2.

DETAILED DESCRIPTION

Described herein are assisted reproductive technology methods, e.g.,methods for treating infertility. In particular, described herein arecontrolled ovarian stimulation (COS) methods that may be particularlyuseful for women at risk of a high ovarian response to controlledovarian stimulation. In some embodiments, the methods are useful foroptimizing ovarian response to COS, improving embryo quality, increasingthe proportion of euploid blastocysts produced by in vitro fertilizationafter controlled ovarian stimulation, decreasing the proportion ofaneuploid blastocysts produced by in vitro fertilization aftercontrolled ovarian stimulation, increasing ongoing pregnancy ratesand/or increasing live birth rates.

The present invention is based on the unexpected finding by theinventors that the use of highly purified menotropin (HP-hMG) inpotential high ovarian responders undergoing controlled ovarianstimulation (COS) may optimize ovarian response, improve embryo quality,increase the proportion of euploid blastocysts, decrease the proportionof aneuploid blastocysts, and improve ongoing pregnancy rates, andincreases live birth rates.

Definitions

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art of assisted reproductivetechnology to which the present invention pertains, unless otherwisedefined. Reference is made herein to various methodologies known tothose of ordinary skill in the art. Any suitable materials and/ormethods known to those of ordinary skill in the art can be utilized incarrying out the present invention. However, specific materials andmethods are described. Materials, reagents and the like to whichreference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” designate boththe singular and the plural, unless expressly stated to designate thesingular only.

As used herein, the term “about” means that the number or range is notlimited to the exact number or range set forth, but encompass rangesaround the recited number or range as will be understood by persons ofordinary skill in the art depending on the context in which the numberor range is used. Unless otherwise apparent from the context orconvention in the art, “about” mean up to plus or minus 10% of theparticular term.

As used herein, “optimizing ovarian response” refers to optimizingovarian response to COS to achieve an optimal number of developingfollicles, such as from 4-15 developing follicles, about 8-15 developingfollicles, about 8-14 developing follicles, or about 11 developingfollicles.

As used herein, “improving embryo quality” refers to increasing theproportion of euploid blastocysts (e.g., blastocysts having the correctnumber of chromosomes) and/or decreasing the proportion of aneuploidblastocysts (e.g., blastocysts having an incorrect number ofchromosomes) produced in a single cycle of ART that involves COSfollowed by in vitro fertilization.

As used herein “ongoing pregnancy” refers to pregnancy with a viablefetus and detectable fetal heartbeat at 10-11 weeks gestation, e.g., at8-9 weeks post blastocyst/embryo transfer.

As used herein “clinical pregnancy” refers to gestation and a detectablefetal heartbeat at 5-6 weeks gestation, e.g., at 3-4 weeks postblastocyst/embryo transfer.

As used herein, “woman” refers to an adult female human. In someembodiments, a woman treated in accordance with the methods describedherein is 35 years old or younger, or is 34 years old or younger. Insome embodiments, a woman treated in accordance with the methodsdescribed herein is 21-38 years old, or 21-37, or 21-36, or 21-34, or21-32, or 21-30 years old. In some embodiments, a woman treated inaccordance with the methods described herein has a BMI of 38 kg/m² orless, 36 kg/m² or less, 34 kg/m² or less, 32 kg/m² or less, 30 kg/m² orless, or 28 kg/m² or less, such as BMI of 18-38, 18-36, 18-34, 18-32,18-30, or 18-28 kg/m². In some embodiments, a woman treated inaccordance with the methods described herein has a BMI of 18-25 kg/m²,18-26 kg/m², 18-29 kg/m², 18-30 kg/m², 18-32 kg/m², 18-34 kg/m², 18-36kg/m², or 18-38 kg/m². In some embodiments, a woman treated inaccordance with the methods described herein is an ovulatory woman,i.e., is not anovulatory. In some embodiments, a woman treated inaccordance with the methods described herein is an ovulatory woman withmenstrual cycles of no greater than 45 days.

As used herein, subjects classified as being “at risk of a high ovarianresponse to controlled ovarian stimulation” refers to women who arelikely to develop high numbers of follicles or oocytes following astandard protocol of controlled ovarian stimulation (COS), such as womenwith a greater than average likelihood of producing 15 or more oocytes.Women may be identified as being at risk if they have generated 15 ormore oocytes in a previous ART cycle, e.g., in a previous COS treatment.Additionally or alternatively, women may be identified as being at riskif they are considered to be at risk of developing OHSS. Additionally oralternatively, women may be identified as being at risk if they have aserum level of anti-Müllerian hormone (AMH) of greater than or equal toabout 5.0 ng/ml (such as greater than or equal to about 5.0 ng/ml) whenassessed as described herein below (or an equivalent level when assessedby a different method). Additionally or alternatively, women may beidentified as being at risk if they have an antral follicle count (AFC)of greater than or equal to 15, greater than or equal to 18, or greaterthan or equal to 20, in both ovaries combined, prior to stimulation.

The term “menotropin” as used herein includes human menopausalgonadotropin or hMG. The term “highly purified menotropin” as usedherein includes HP-hMG available under the trademark MENOPUR® fromFerring B. V., that contains both follicle stimulating hormone (FSH) andhuman chorionic gonadotrophin (hCG)-driven luteinizing hormone(LH)-activity.

The term “GnRH agonist” as used herein includes gonadotropin-releasinghormone (GnRH) agonists such as buserelin (e.g., Suprecur®), leuprorelin(e.g., Lupron®), nafarelin (e.g., Synarel®), and triptorelin (e.g.,Trelstar®).

The term “GnRH antagonist” as used herein includesgonadotropin-releasing hormone (GnRH) antagonists, such as ganirelixacetate (e.g., Orgalutran®) and cetrorelix acetate (e.g., Cetrotide®),which block the action of GnRH by competitive blocking of the GnRHreceptors on pituitary gonadotropes, and thus prevent gonadotropinproduction and eggs premature release.

As used herein, the phrase “effective amount” refers to the dosage thatprovides the specific pharmacological effect for which the drug isadministered in a subject in need of such treatment. It is emphasizedthat a therapeutically effective amount will not always be effective intreating the conditions described herein, even though such dosage isdeemed to be a therapeutically effective amount by those of skill in theart. For convenience only, exemplary dosages and therapeuticallyeffective amounts are provided below with reference to adult femalehuman subjects. Those skilled in the art can adjust such amounts inaccordance with standard practices as needed to treat a specific subjectand/or condition/disease.

As noted above, the present invention provides reproductive technologymethods that involve using highly purified menotropin (HP-hMG) in womenat risk of a high ovarian response to COS and undergoing COS. As alsonoted above, for the purposes of the methods disclosed herein, women maybe identified as being at risk of a high ovarian response to COS if theyhave a serum level of anti-Müllerian hormone (AMH) of greater than orequal to about 5.0 ng/ml, such as greater than or equal to 5.0±0.5ng/ml, such as greater than or equal to 5.2±0.5 ng/ml, such as greaterthan or equal to about 5.0±0.5 ng/ml, such as greater than or equal toabout 5.2±0.5 ng/ml, when measured using a Beckmann-Coulter Gen 2 assayas described in Arce et al., Fertility and Sterility 99: 1644-53 (2013),or an equivalent AMH level assessed by a different method (± itscoefficient of variability). Serum levels of AMH are a surrogate markerfor functional ovarian follicle reserve, and a positive correlationbetween serum levels of AMH and ovarian response (e.g., oocyte yield)have been reported. Id. However, according to Arce et al., AMH levelshave not been correlated with embryo quality. Id.

Additionally or alternatively, as also noted above, for the purposes ofthe methods disclosed herein, women may be identified as being at riskof a high ovarian response to COS if they have an antral follicle count(AFC) of greater than or equal to 15, greater than or equal to 18, orgreater than or equal to 20, in both ovaries combined, prior tostimulation. Antral follicles are small follicles (about 2-8 mm indiameter) that can be visualized and measured via transvaginalultrasound. Antral follicles are another marker for ovarian reserve, andAFC has been used (along with other markers) to predict ovarian responseto COS, e.g., to predict whether a given subject will be a low, normal,or high responder. However, AFC has not been correlated with embryoquality.

Assisted Reproductive Technology Methods

The methods described herein are useful in any reproductive technologymethods that involve controlled ovarian stimulation (COS), such as forin vitro fertilization, including intra-cytoplasmic sperm injection(ICSI), including methods involving fresh transfer of fertilized eggs(e.g., blastocysts/embryos) and methods that involving freezingfertilized eggs (e.g., blastocysts/embryos) for later implantation.

In specific embodiments, the subject undergoing COS is at risk of a highovarian response to COS. In specific embodiments, the subject isidentified by having a serum level of AMH of greater than or equal toabout 5.0 ng/ml, such as greater than or equal to 5.0±0.5 ng/ml, such asgreater than or equal to about 5.0±0.5 ng/ml ng/ml, such as greater thanor equal to 5.2±0.5 ng/ml, such as greater than or equal to about5.2±0.5 ng/ml ng/ml, when measured using a Beckmann-Coulter Gen 2 assayas described in Arce et al., Fertility and Sterility 99: 1644-53 (2013),or an equivalent AMH level assessed by a different method (± itscoefficient of variability).

In some embodiments, the subject is additionally or alternativelyidentified by having an AFC of greater than or equal to 15, greater thanor equal to 18, or greater than or equal to 20, in both ovariescombined, prior to stimulation. In some embodiments, the subject isexcluded from treatment by the methods described herein if the subjecthas an AFC of less than 8, or less than 10, in both ovaries combined,prior to stimulation. In some embodiments, subjects treated by themethods described herein have an AFC of at least 10 (≥10) in bothovaries combined prior to stimulation.

The subject may be additionally or alternatively identified as being atrisk if they have generated 15 or more oocytes in a previous ART cycle,e.g., in a previous COS treatment, and/or if they are considered to beat risk of developing OHSS.

In specific embodiments, the methods include administering HP-hMG to thesubject in an amount effective to stimulate follicle development. Insome embodiments, HP-hMG is administered at a dose of from about 75 IUor 100 IU to about 300 IU/day, or from about 100 to about 200 IU/day,for from about 1 to about 20 days, such as for at least 5 days.Pharmaceutical compositions comprising HP-hMG are availablecommercially, such as the MENOPUR® product sold by Ferring B. V., whichis formulated for subcutaneous injection. Typically, a starting dose is150 IU/day for the first 5 days, which may be adjusted thereafter (e.g.,on day 6 onward) based on the subject's ovarian (follicular) response,which may be assessed, for example, by transvaginal ultrasound (TVUS).For example, once the lead follicle reaches >12 mm in diameter, the dosecan be adjusted by 75 IU per adjustment. Typically, the dose is adjustedup or down when both the serum estradiol level and the number offollicles>12 mm are either too low or too high on the 6 day ofstimulation. HP-hMG is administered daily until the desired level offollicle production is reached. For example, HP-hMG may be administereduntil three follicles have developed with a diameter of >17 mm, as maybe determined by TVUS. Typically, the maximum HP-hMG dosing period is 20days.

In some embodiments, the methods include the administration of a GnRHantagonist during a portion of the period of HP-hMG administration. Forexample, a GnRH antagonist may be administered once the lead folliclereaches 14 mm in diameter, and continued through the remainder of theperiod of HP-hMG administration. When the GnRH antagonist is ganirelixacetate (such as Orgalutran®), a typical dose is 0.25 mg/dayadministered subcutaneously.

In other embodiments, the methods include the administration of a GnRHagonist prior to ovarian stimulation, such as the administration oftriptorelin (typically at 0.1 mg/day subcutaneously) or leuprorelin(e.g., Lupron®).

Once the desired level of follicle production is reached, ovulation canbe stimulated by methods known in the art, such as by a bolus injectionof human chorionic gonadotropin

(hCG). A typical dose of recombinant hCG (such as Ovitrelle®) is 250 μg(6,500 IU of hCG activity), usually administered by a singlesubcutaneous injection. Then, oocytes are retrieved and fertilized bymethods known in the art, such as ICSI.

For fresh transfer methods, one or more blastocysts are selected fortransfer a few days after fertilization (e.g. after 5 days). Remainingblastocysts can be frozen by methods known in the art for futuretransfer (including vitrification). In specific embodiments, the methodsdescribed herein are used in a single blastocyst transfer protocol,wherein a single blastocyst is selected for fresh transfer. Inaccordance with those embodiments, remaining blastocysts can be frozenby methods known in the art for future transfer.

For “freeze all” methods, selected blastocysts are frozen by methodsknown in the art for future transfer.

In specific embodiments, blastocyst selection is guided by morphologicalanalysis. For example, morphological blastocyst quality can be assessedby determining blastocyst expansion and hatching status, inner cell massgrading and trophectoderm grading according to the Gardner blastocystgrading system. See, e.g., Van den Abbeel et al., Reproductive Biomed.OnLine 27: 353-61 (2013). For example, blastocysts with blastocystexpansion and hatching status 4, 5 or 6, inner cell mass grading A, andtrophectoderm grading A or B may be deemed of “excellent” quality, whileblastocysts with blastocyst expansion and hatching status 3, 4, 5 or 6,inner cell mass grading A or B, and trophectoderm grading A or B may bedeemed of “good” quality.

Additionally or alternatively, blastocyst quality is assessed bychromosomal analysis, including genetic screening (e.g., preimplantationgenetic screening, or PGS) to detect aneuploidy. In some embodiments,chromosomal analysis includes comprehensive chromosomal screening (CCS)as described, for example, in Forman et al., Fertility and Sterility100: 718-24 (2013). As reported in this paper, CCS can be conductedprior to fresh transfer or prior to blastocyst freezing for futuretransfer.

In some embodiments, luteal phase support is provided by theadministration of progesterone, such as vaginal progesterone inserts,from the day after oocyte retrieval to 10-15 days afterblastocyst/embryo transfer, and/or from the day of blastocyst/embryotransfer to the confirmation of ongoing pregnancy, in accordance withprotocols known in the art.

In some embodiments, clinical pregnancy is confirmed by TVUS at 5-6weeks gestation (e.g., 3-4 weeks after blastocyst/embryo transfer). Insome embodiments, ongoing pregnancy is confirmed by TVUS at 10-11 weeksgestation (e.g., 8-9 weeks after blastocyst/embryo transfer).

In some embodiments, the subject is followed to assess pregnancy outcome(e.g. live birth) and/or neonatal health.

As set forth above, the methods described herein are useful forimproving embryo quality, increasing the proportion of euploidblastocysts, decreasing the proportion of aneuploid blastocysts,increasing ongoing pregnancy rates and/or increasing live birth rates,as compared to comparable methods using recombinant follicle stimulatinghormone (rFSH) as the gonadotropin. In particular, the methods describedherein may result in an increased proportion of euploid blastocysts(e.g., a decreased proportion of aneuploidy blastocysts), as compared tocomparable methods using rFSH as the gonadotropin. The methods describedherein also may result in increased ongoing pregnancy rates and/orincreased live birth rates, as compared to comparable methods using rFSHas the gonadotropin, such as an 8% or 10% or greater increase in ongoingpregnancy rates and/or increased live birth rates. In some embodiments,the methods result in fewer eggs but a higher proportion of euploidblastocysts as compared to comparable methods using rFSH as thegonadotropin.

The following exemplary methods are encompassed by the foregoingdisclosure:

A method for improving the quality of an embryo produced by in vitrofertilization after controlled ovarian stimulation in a woman at risk ofa high ovarian response thereto, comprising selecting a woman at risk ofa high ovarian response to controlled ovarian stimulation having a serumanti-Müllerian hormone (AMH) level greater than or equal to 5.0±0.5ng/ml, when measured using a Beckmann-Coulter Gen 2 assay or acomparable AMH level measured by a different method; and administeringto the selected woman an amount of highly purified menotropin (HP-hMG)effective to stimulate follicle development.

A method for improving the quality of an embryo produced by in vitrofertilization after controlled ovarian stimulation in a woman at risk ofa high ovarian response thereto, comprising administering an amount ofhighly purified menotropin (HP-hMG) effective to stimulate follicledevelopment to a woman selected as being at risk of a high ovarianresponse to controlled ovarian stimulation, wherein the selected womanhas a serum anti-Müllerian hormone (AMH) level greater than or equal to5.0±0.5 ng/ml, when measured using a Beckmann-Coulter Gen 2 assay or acomparable AMH level measured by a different method.

A method for increasing the proportion of euploid blastocysts producedby in vitro fertilization after controlled ovarian stimulation in awoman at risk of a high ovarian response thereto, comprising selecting awoman at risk of a high ovarian response to controlled ovarianstimulation having a serum anti-Müllerian hormone (AMH) level greaterthan or equal to 5.0±0.5 ng/ml when measured using a Beckmann-CoulterGen 2 assay or a comparable AMH level measured by a different method;and administering to the selected woman an amount of highly purifiedmenotropin (HP-hMG) effective to stimulate follicle development.

A method for increasing the proportion of euploid blastocysts producedby in vitro fertilization after controlled ovarian stimulation in awoman at risk of a high ovarian response thereto, comprisingadministering an amount of highly purified menotropin (HP-hMG) effectiveto stimulate follicle development to a woman selected as being at riskof a high response to controlled ovarian stimulation, wherein theselected woman has a serum anti-Müllerian hormone (AMH) level greaterthan or equal to 5.0±0.5 ng/ml when measured using a Beckmann-CoulterGen 2 assay or a comparable AMH level measured by a different method.

A method for treating infertility in a woman at risk of a high ovarianresponse to controlled ovarian stimulation, comprising selecting a womanat risk of a high ovarian response to controlled ovarian stimulationhaving (i) a serum anti-Müllerian hormone (AMH) level greater than orequal to 5.0±0.5 ng/ml when measured using a Beckmann-Coulter Gen 2assay or a comparable AMH level measured by a different method; andadministering to the selected woman an amount of highly purifiedmenotropin (HP-hMG) effective to stimulate follicle development.

A method for treating infertility in a woman at risk of a high ovarianresponse to controlled ovarian stimulation, comprising administering anamount of highly purified menotropin (HP-hMG) effective to stimulatefollicle development to a woman selected as being at risk of a highovarian response to controlled ovarian stimulation, wherein the selectedwoman has a serum anti-Müllerian hormone (AMH) level greater than orequal to 5.0±0.5 ng/ml when measured using a Beckmann-Coulter Gen 2assay or a comparable AMH level measured by a different method.

A method for effecting controlled ovarian stimulation in a woman at riskof a high ovarian response thereto, comprising selecting a woman at riskof a high ovarian response to controlled ovarian stimulation having aserum anti-Müllerian hormone (AMH) level greater than or equal to5.0±0.5 ng/ml when measured using a Beckmann-Coulter Gen 2 assay or acomparable AMH level measured by a different method; and administeringto the selected woman an amount of highly purified menotropin (HP-hMG)effective to stimulate follicle development.

A method for effecting controlled ovarian stimulation in a woman at riskof a high ovarian response thereto, comprising administering an amountof highly purified menotropin (HP-hMG) effective to stimulate follicledevelopment to a woman selected as being at risk of a high ovarianresponse to controlled ovarian stimulation, wherein the selected womanhas a serum anti-Müllerian hormone (AMH) level greater than or equal to5.0±0.5 ng/ml when measured using a Beckmann-Coulter Gen 2 assay or acomparable AMH level measured by a different method.

A method for increasing ongoing pregnancy rates in a woman at risk of ahigh ovarian response to controlled ovarian stimulation, comprisingselecting a woman at risk of a high ovarian response to controlledovarian stimulation having a serum anti-Müllerian hormone (AMH) levelgreater than or equal to 5.0±0.5 ng/ml when measured using aBeckmann-Coulter Gen 2 assay or a comparable AMH level measured by adifferent method; and administering to the selected woman an amount ofhighly purified menotropin (HP-hMG) effective to stimulate follicledevelopment.

A method for increasing ongoing pregnancy rates in a woman at risk of ahigh ovarian response to controlled ovarian stimulation, comprisingadministering an amount of highly purified menotropin (HP-hMG) effectiveto stimulate follicle development to a woman selected as being at riskof a high ovarian response to controlled ovarian stimulation, whereinthe selected woman has a serum anti-Müllerian hormone (AMH) levelgreater than or equal to 5.0±0.5 ng/ml when measured using aBeckmann-Coulter Gen 2 assay or a comparable AMH level measured by adifferent method.

A method for increasing live birth rates in a woman at risk of a highovarian response to controlled ovarian stimulation, comprising selectinga woman at risk of a high ovarian response to controlled ovarianstimulation having a serum anti-Müllerian hormone (AMH) level greaterthan or equal to 5.0±0.5 ng/ml when measured using a Beckmann-CoulterGen 2 assay or a comparable AMH level measured by a different method;and administering to the selected woman an amount of highly purifiedmenotropin (HP-hMG) effective to stimulate follicle development.

A method for increasing live birth rates in a woman at risk of a highovarian response to controlled ovarian stimulation, comprisingadministering an amount of highly purified menotropin (HP-hMG) effectiveto stimulate follicle development to a woman selected as being at riskof a high ovarian response to controlled ovarian stimulation, whereinthe selected woman has a serum anti-Müllerian hormone (AMH) levelgreater than or equal to 5.0±0.5 ng/ml when measured using aBeckmann-Coulter Gen 2 assay or a comparable AMH level measured by adifferent method.

A method for promoting the development of euploid blastocysts in a womanat risk of a high ovarian response to controlled ovarian stimulation,comprising selecting a woman at risk of a high ovarian response tocontrolled ovarian stimulation having a serum anti-Müllerian hormone(AMH) level greater than or equal to 5.0±0.5 ng/ml when measured using aBeckmann-Coulter Gen 2 assay or a comparable AMH level measured by adifferent method; and administering to the selected woman an amount ofhighly purified menotropin (HP-hMG) effective to stimulate follicledevelopment.

A method for promoting the development of euploid blastocysts in a womanat risk of a high ovarian response to controlled ovarian stimulation,comprising administering an amount of highly purified menotropin(HP-hMG) effective to stimulate follicle development to a woman selectedas being at risk of a high ovarian response to controlled ovarianstimulation, wherein the selected woman has a serum anti-Müllerianhormone (AMH) level greater than or equal to 5.0±0.5 ng/ml when measuredusing a Beckmann-Coulter Gen 2 assay or a comparable AMH level measuredby a different method.

In any of these methods, the woman may have an antral follicle count(AFC) of greater than or equal to 10 in both ovaries combined, prior tostimulation.

In any of these methods, the woman may have an antral follicle count(AFC) of greater than or equal to 15 in both ovaries combined, prior tostimulation.

Any of these methods may result in an increased proportion of euploidblastocysts as compared to a comparable method using recombinantfollicle-stimulating hormone (rFSH) as the gonadotropin, and/or anincreased ongoing pregnancy rate, and/or an increased live birth rate.

In any of these methods, the amount of HP-hMG administered may be from100-300 IU per day for from about 1 to about 20 days, or from 100-200 IUper day for from about 1 to about 20 days.

In any of these methods, the HP-hMG may be administered for at least 5days.

Any of these methods may further comprise administering a gonadotropinhormone releasing hormone (GnRH) agonist, and/or a GnRH antagonist.

Any of these methods may further comprise administering an amount of hCGeffective to trigger ovulation, harvesting oocytes from the woman, invitro fertilization of harvested oocytes (such as intra-cytoplasmicsperm injection (ICSI)), assessing the chromosomal quality ofblastocysts obtained from the woman after in vitro fertilization ofoocytes harvested from the woman, blastocyst transfer of a blastocystdetermined to be a euploid blastocyst (such as fresh transfer), and/orfreezing a blastocyst determined to be a euploid blastocyst.

Further aspects of the methods described herein are illustrated in thefollowing examples, which are not limiting in any respect.

EXAMPLES Example 1—Retrospective Analysis

A retrospective analysis was undertaken of data collected in tworandomized controlled clinical trials comparing treatment outcome inpatients undergoing stimulation with HP-hMG or recombinant FSH followinga long GnRH agonist protocol (Anckaert et al., Human Reproduction 27:1829-39 (2012)) or a GnRH antagonist protocol (Arce el al., Fertilityand Sterility, 99: 1644-53 (2013).

1. Study Populations

The main inclusion criteria for the long agonist trial were women aged21-37 years with major indications for IVF (such as tubal factorinfertility, unexplained infertility or mild male factor infertility),FSH levels within normal limits (1-12 IU/L), BMI of 18-29, and regularmenstrual cycles of 21-35 days which were presumed to be ovulatory. Themain inclusion criteria for the antagonist trial were women aged 21-34years with a primary infertility diagnosis of unexplained infertility ormild male factor infertility, FSH levels of 1-12 IU/L, BMI of 18-25, andregular menstrual cycles of 24-35 days,

2. Study Protocols

In the long agonist protocol, down-regulation was performed usingtriptorelin (0.1 mg/day) (Decapeptyl®, Ferring Pharmaceuticals A/S)initiated 5-7 days before the estimated start of next menses andcontinued until the end of gonadotropin administration. Gonadotropin(HP-hMG or rFSH) was administered at 225 IU/day for the first 5 days,and then adjusted according to ovarian response.

In the antagonist protocol, gonadotropin (HP-hMG or rFSH) wasadministered at 150 IU/day for the first 5 days, and then adjustedaccording to ovarian response from day 6. On day 6 GnRH antagonist(ganirelix, Orgalutran, MSD) was initiated at 0.25 mg/day and continuedthroughout gonadotropin-treatment.

In both protocols, 250 μg hCG (Choriogonadotropin alpha, Ovitrelle,Merck Serono) was administered when three follicles of ≥17 mm diameterwere observed, and oocyte retrieval took place 36±2 hours later.

Luteal support was provided by vaginal administration of progesterone

In the long agonist protocol, 1 or 2 embryos were transferred on day 3.In the antagonist protocol, 1 blastocyst was transferred on day 5.

Live birth was defined as delivery of (at least) one live-born neonate.

3. Serum Assays

AMH was analyzed by enzyme-linked immunosorbent assay (long agonisttrial: Immunotech Beckman Coulter AMH ELISA; antagonist trial: BeckmanCoulter Gen 2 ELISA; 1 ng/ml=7.14 pmol/L). The AMH assays had asensitivity of 0.35 and 0.08 ng/ml and total imprecision (% coefficientof variation) of <9.5 and <7.7 in Immunotech Beckman Coulter and BeckmanCoulter Gen 2, respectively. FSH, estradiol and progesterone wereanalyzed by electrochemiluminescence immunoassay (Roche-DiagnosticsECLIA).

4. Statistical Analysis

In total, the two trials comprised 1372 women. In the retrospectiveanalysis, women were classified as potential high-responders if initialAMH was in the uppermost quartile (75^(th) percentile) of the observedAMH distribution. In both protocols, the 75^(th) percentile wasidentical (5.2 ng/ml=37.4 pmol/l). In the retrospective analysis, onehundred fifty-five women treated in the long GnRH agonist protocol (76and 79 in the HP-hMG and rFSH groups, respectively) and 188 women in theGnRH antagonist protocol (87 and 101 in the HP-hMG and rFSH groups,respectively) were classified as potential high-responders.

In each protocol, baseline characteristics, end-of-stimulation data,ovarian response and embryo data were compared between the women groupedaccording to their AMH value on stimulation day 1 (>75th versus≥75thpercentile). Similar analyses were performed for the potentialhigh-responders comparing gonadotropin treatments (HP-hMG versus rFSH)within each protocol. Continuous and categorical data were comparedusing the Wilcoxon test and the Chi-Square or Fisher's exact test,respectively. For the potential high-responders, risk of high response(≥15 oocytes retrieved) and chance of live birth were compared betweentreatments using the Chi-square test. The observed differences in livebirth rates between gonadotropin-treatment groups were further analyzedin the pooled population of potential high-responders from bothprotocols to determine if they could be attributed to baselinecharacteristics or end-of-stimulation variables. For each variable, alogistic regression model was fitted including treatment group and thevariable in question in the linear predictor. Only fresh treatmentcycles were included in the present dataset.

5. Results

High AMH Category Versus Non-High AMH Category

In both the long agonist protocol and the antagonist protocol, the womenin the high AMH category were characterized by younger age, longermenstrual cycle length, higher AFC, lower FSH and larger ovarian volumeat start of stimulation than women in the non-high AMH category (p≤0.003for each variable). These features are shown in Table 1.

TABLE 1 Demographics and baseline, end-of-stimulation, oocyte and embryodata of the women grouped by the AMH concentration at start ofstimulation (quartiles 1-3 versus quartile 4). Long GnRH agonistprotocol GnRH antagonist protocol AMH Q1-Q3 ≤ 75th AMH Q4 > 75th AMHQ1-Q3 ≤ 75th AMH Q4 > 75th (≤5.2 ng/ml) (>5.2 ng/ml) (≤5.2 ng/ml) (>5.2ng/ml) Variable (n = 468) (n = 155) p Value* (n = 561) (n = 188) pValue* Clinical characteristics Age (years) 31 (29, 34) 30 (28, 32)<0.001 31 (29, 33) 30 (28, 32) <0.001 BMI (kg/m²) 21.9 (20.3, 24.0) 21.3(20.1, 23.5) 0.113 21.9 (20.3, 23.8) 21.8 (20.5, 23.5) 0.868 Cyclelength (days) 28 (28, 29) 29 (28, 30) <0.001 28 (28, 29) 29 (28, 30)<0.001 First treatment cycle, n (%) 327 (70%) 104 (67%) 0.517 427 (76%)134 (71%) 0.186 Day 1 (before stimulation) Ovarian volume (ml) 8.5 (6.0,11.8) 10.4 (7.7, 14.3) <0.001 10.6 (8.0, 14.2) 13.2 (9.6, 16.8) <0.001AFC (n) 10 (7, 14) 11 (8, 18) <0.001 14 (11, 17) 18 (15, 22) <0.001 AMH(ng/ml) 3.0 (2.1, 4.0) 7.0 (5.8, 8.5) <0.001 2.4 (1.4, 3.6) 6.9 (6.0,8.7) <0.001 FSH (IU/l) 3.8 (3.0, 4.9) 3.4 (2.7, 4.4) 0.003 7.2 (6.2,8.5) 6.5 (5.7, 7.6) <0.001 End-of-stimulation Estradiol (nmol/l) 5.5(4.0, 7.3) 8.5 (6.2, 13.0) <0.001 5.7 (4.1, 8.2) 8.7 (6.3, 13.3) <0.001Progesterone (nmol/l) 2.6 (2.0, 3.4) 3.2 (2.4, 3.9) <0.001 2.5 (1.9,3.2) 2.9 (2.1, 3.8) <0.001 Progesterone/estradiol ratio 0.46 (0.35,0.63) 0.36 (0.24, 0.49) <0.001 0.42 (0.30, 0.60) 0.32 (0.21, 0.44)<0.001 Follicles ≥12 mm (n) 10 (8, 13) 15 (12, 19) <0.001 10 (7, 13) 15(11, 18) <0.001 Endometrial thickness (mm) 11 (9, 12) 11 (10, 12) 0.07910 (9, 12) 11 (10, 12) 0.039 Endometrial echogenicity pattern 39, 49, 1334, 51, 15 0.544 40, 51, 9 36, 54, 10 0.668 (hypo, iso, hyper) (%) Cyclecancellation for 2 (<1%) 7 (5%) 0.001 1 (<1%) 1 (<1%) 0.439 ovarianhyper-response, n (%) Early OHSS (moderate/severe), n (%) 1 (<1%) 7 (5%)<0.001 4 (<1%) 8 (4%) 0.003 Intervention for ovarian 2 (<1%) 11 (7%)<0.001 16 (3%) 19 (10%) <0.001 hyper-response, n (%) Oocyte retrievalWomen with oocyte 446 (95%) 145 (94%) 0.392 537 (96%) 185 (98%) 0.088retrieval, n (%) Oocytes retrieved (n) 9 (6, 12) 14 (10, 18) <0.001 8(5, 11) 12 (9, 17) <0.001 Women with ≥15 oocytes 76 (16%) 65 (42%)<0.001 63 (11%) 76 (40%) <0.001 retrieved, n (%) Fertilization andembryo data Fertilisation rate (%) 60 (33, 75) 52 (29, 70) 0.091 60 (43,75) 58 (42, 71) 0.193 Embryos, day 3 (n) 2 (1, 5) 3 (2, 6) 0.029 Womenwith top-quality 199 (45%) 73 (50%) 0.229 embryo(s), day 3, n (%)†Blastocysts, day 5 (n) 2 (1, 4) 3 (1, 6) <0.001 Women with good-quality266 (50%) 106 (57%) 0.068 blastocyst(s), day 5, n (%)‡ Women withtransfer, n (%)¶ 397 (89%) 122 (84%) 0.119 462 (86%) 159 (86%) 0.976Values are median (IQR) unless otherwise indicated. *Wilcoxon test(continuous data); Chi-Square test or Fisher's exact test (categorialdata). †Top-quality embryos were defined as 4-5 cells on day 2, ≥7 cellson day 3, equally-sized blastomeres and ≤20% fragmentation on day 3 andno multinucleation. ‡Good-quality blastocysts were defined asblastocysts with expansion and hatching score ≥4 and with inner cellmass and trophectoderm grades of A or B, using the definitions describedby Gardner & Schoolcraft, “In-vitro culture of human blastocysts,” InTOWARDS REPRODUCTIVE CERTAINTY: FERTILITY AND GENETICS BEYOND 1998,Jansen & Mortimer, eds. (The Parthenon Publishing Group, New York 1999),pg. 378-88. Among women with oocytes retrieved.

The retrospective analysis revealed that, independent of the protocolused, women with high AMH exhibited significantly (p<0.001 for eachvariable) higher serum levels of estradiol and progesterone as well asincreased number of growing follicles≥12 mm at end of stimulation.Further, the women with high AMH had significantly (p≤0.003 for eachvariable) more oocytes retrieved, increased occurrence of high response,higher frequency of early OHSS and interventions for hyper-response. Inthe long agonist protocol, cycle cancellation due to ovarianhyper-response occurred more frequently among women in the high AMHcategory (p=0.001). At end of stimulation, no clinically relevantdifferences were noted in endometrial thickness or echogenicity patternsbetween the two AMH categories. Significantly more embryos on day 3(long agonist protocol: p=0.029) or blastocysts on day 5 (antagonistprotocol: p<0.001) were available in women with high AMH, but theproportion of women with top-quality embryo(s) or good-qualityblastocyst(s) were similar in the two AMH categories.

6. HP-hMG Versus rFSH Stimulation in High AMH Category

Within each protocol, there were no clinically relevant differencesbetween the two gonadotropin-treatment groups in the high AMH categoryregarding demographics, fertility history and markers of ovarianreserve, as shown in Table 2 below. BMI was significantly lower inrFSH-treated women, but was not believed to be of clinical relevance.(However, it is possible that a higher BMI could blunt ovarianresponse.) At the end of stimulation, higher estradiol levels (p=0.012)and lower progesterone levels (p<0.001) were observed with HP-hMG in theantagonist and long agonist protocol, respectively.

TABLE 2 Comparison of baseline, end-of-stimulation, oocyte and embryocharacteristics between HP-hMG- and rFSH-treated women with potentialfor being high-responders by a high AMH at start of stimulation. LongGnRH agonist protocol GnRH antagonist protocol AMH Q4: >75th (>5.2ng/ml) AMH Q4: >75th (>5.2 ng/ml HP-hMG rFSH HP-hMG rFSH Variable (n =76) (n = 79) p Value * (n = 87) (n = 101) p Value * Clinicalcharacteristics Age (years) 30 (28, 32) 30 (28, 32) 0.743 30 (28, 33) 30(28, 31) 0.039 BMI (kg/m²) 22.5 (20.7, 23.8) 20.8 (19.8, 22.8) 0.00222.1 (21.0, 23.9) 21.6 (20.1, 23.0) 0.022 Cycle length (days) 29 (28,30) 29 (28, 30) 0.682 29 (28, 30) 29 (28, 31) 0.382 First treatmentcycle, n (%) 52 (68%) 52 (66%) 0.731 57 (66%) 77 (76%) 0.105 Day 1(before start of stimulation) Ovarian volume (ml) 10.3 (7.9, 13.9) 10.5(7.7, 14.8) 0.807 13.4 (9.1, 17.0) 13.0 (9.9, 16.7) 0.885 AFC (n) 12 (8,20) 11 (8, 16) 0.486 18 (15, 22) 18 (15, 22) 0.934 AMH (ng/ml) 7.0 (5.9,8.5) 7.0 (5.7, 8.4) 0.912 7.1 (6.2, 8.7) 6.8 (6.0, 8.3) 0.347 FSH (IU/1)3.2 (2.6, 4.4) 3.6 (2.8, 4.4) 0.257 6.7 (5.6, 7.7) 6.4 (5.7, 7.5) 0.251End-of-stimulation Estradiol (nmol/1) 8.7 (6.4, 13.0) 8.4 (6.1, 12.8)0.736 9.7 (6.8, 14.8) 7.8 (5.5, 12.4) 0.012 Progesterone (nmol/1) 2.7(1.9, 3.6) 3.6 (2.8, 4.5) <0.001 2.8 (2.1, 3.9) 3.0 (2.1, 3.8) 0.857Progesterone/estradiol ratio 0.31 (0.21, 0.42) 0.43 (0.30, 0.52) <0.0010.26 (0.20, 0.41) 0.34 (0.24, 0.47) 0.011 Follicles ≥12 mm (n) 15 (12,18) 16 (13, 19) 0.274 14 (11, 18) 16 (12, 19) 0.064 Endometrialthickness (mm) 11 (10, 12) 11 (10, 12) 0.522 11 (10, 12) 11 (10, 12)0.478 Endometrial echogenicity pattern 44, 43, 13 24, 60, 17 0.033 31,58, 11 41, 50, 9 0.386 (hypo, iso, hyper) (%) Cycle cancellation forovarian 3 (4%) 4 (5%) 1.000 0 (0%) 1 (<1%) — hyper-response, n (%) EarlyOHSS (moderate/severe), 3 (4%) 4 (5%) 1.000 3 (3%) 5 (5%) 0.727 n (%)Intervention for ovarian 5 (7%) 6 (8%) 1.000 7 (8%) 12 (12%) 0.470hyper-response, n (%) Oocyte retrieval Women with oocyte 72 (95%) 73(92%) 0.555 85 (98%) 100 (99%) 0.475 retrieval, n (%) Oocytes retrieved(n) 12 (9, 16) 15 (11, 20) 0.007 12 (8, 15) 14 (10, 19) 0.033 Women with≥15 oocytes 25 (33%) 40 (51%) 0.025 27 (31%) 49 (49%) 0.015 retrieved, n(%) Fertilisation and embryo data Fertilisation rate (%) 50 (27, 73) 56(35, 69) 0.826 57 (43, 69) 60 (41, 73) 0.663 Embryos on day 3 (n) 3 (2,6) 4 (2, 6) 0.806 Women with top-quality 38 (53%) 35 (48%) 0.561embryo(s) on day 3, n (%)† Blastocysts on day 5 (n) 3 (1, 6) 3 (1, 6)0.969 Women with good-quality 55 (65%) 51 (51%) 0.060 blastocyst(s) onday 5, n (%)‡ Women with transfer, n (%)¶ 61 (85%) 61 (84%) 0.848 72(85%) 87 (87%) 0.655 Values are median (IQR) unless otherwiseindicated. * Wilcoxon test (continuous data); Chi-Square test orFisher's exact test (categorial data). †Top-quality embryos were definedas 4-5 cells on day 2, ≥7 cells on day 3, equally-sized blastomeres and≤20% fragmentation on day 3 and no multinucleation. ‡Good-qualityblastocysts were defined as blastocysts with expansion and hatchingscore ≥4 and with inner cell mass and trophectoderm grades of A or B,using the definitions described by Gardner & Schoolcraft, supra. Amongwomen with oocytes retrieved.

The retrospective analysis revealed that HP-hMG was associated with alower median number of oocytes retrieved in women with high AMH ascompared with rFSH (long agonist protocol: −3 oocytes, p=0.007;antagonist protocol: −2 oocytes, p=0.033). In both protocols, thepercentage of women with a high ovarian response was significantly lowerfor HP-hMG compared with rFSH (long agonist protocol: 33% versus 51%,p=0.025; antagonist protocol: 31% versus 49%, p=0.015)(see FIG. 1A).Therefore, the risk of high response was reduced with HP-hMG by 35 and37%, respectively. There were no apparent differences between the twogonadotropin groups concerning cycle cancellations due to excessiveresponse, early moderate/severe OHSS or interventions for excessiveresponse in either protocol.

Within each protocol, fertilization rate, number of embryos/blastocystsavailable for transfer, women with top-quality embryo(s)/good-qualityblastocyst(s) and percentages of women with transfer were similarbetween the HP-hMG and rFSH groups in the high AMH category. However, inboth protocols a statistical trend (p<0.10) for improved live birth rateper started cycle was observed for HP-hMG compared with rFSH (see FIG.1B). When restricted to women with embryo transfer, the difference inlive birth rate between HP-hMG and rFSH was statistically significant(p=0.043) in the antagonist protocol.

When the data of women with high AMH from both protocols wereintegrated, HP-hMG treatment was associated with significantly lowerincidence of high response (32% (52/163) versus 49% (89/180), p<0.001)and increased live birth rate per started cycle (34% (55/163) versus 22%(39/180), p=0.012) as well as per embryo transfer cycle (41% (55/133)versus 26% (39/148), p=0.008) compared with rFSH treatment. The logisticregression analysis (Table 3 below) indicated that the type of GnRHprotocol did not explain the difference in live birth rates betweenHP-hMG and rFSH, as it remained significant (p=0.012) in the adjustedanalysis. The probability of a live birth significantly increased withthe availability of a top-quality embryo/good-quality blastocyst fortransfer (p<0.001), while an increased progesterone level (p=0.042) andincreased progesterone/estradiol ratio (p=0.042) at end of stimulationsignificantly decreased the probability of live birth (Table 3 below).However, in all adjusted analyses the difference between the twogonadotropin preparations remained significant (p<0.05) indicating thatthe higher live birth rate in women with high AMH and stimulated withHP-hMG could not be attributed to differences in the baseline andend-of-stimulation variables examined.

TABLE 3 Logistic regression analysis of live birth rate adjusting forbaseline characteristics or end-of-stimulation variables. Overallpopulation of potential high-responders (n = 343) Variables OR [95% CI]P-value^(a) Adjusting for type of stimulation protocol Antagonistprotocol vs. long agonist protocol 1.11 [0.69; 1.80] 0.666 HP-hMG vs.rFSH 1.85 [1.14; 2.99] 0.012 Adjusting for age Age (years) 1.00 [0.92;1.09] 0.975 HP-hMG vs. rFSH 1.84 [1.14; 2.98] 0.013 Adjusting for BMIBMI (kg/m²) 1.03 [0.92; 1.15] 0.579 HP-hMG vs. rFSH 1.79 [1.10; 2.92]0.020 Adjusting for menstrual cycle length Cycle length (days) 1.05[0.92; 1.191 0.500 HP-hMG vs. rFSH 1.84 [1.14; 2.98] 0.013 Adjusting forfirst treatment cycle First treatment cycle 1.09 [0.65; 1.85] 0.737HP-hMG vs. rFSH 1.85 [1.14; 2.991 0.012 Adjusting for total number offollicles on stimulation day 1 Total number of follicles 0.99 [0.96;1.02] 0.435 HP-hMG vs. rFSH 1.89 [1.16; 3.07] 0.010 Adjusting for FSH onstimulation day 1 FSH (IU/l) 1.10 [1.00; 1.21] 0.060 HP-hMG vs. rFSH1.89 [1.16; 3.06] 0.010 Adjusting for estradiol at end-of-stimulationEstradiol (nmol/l) 0.99 [0.96; 1.03] 0.716 HP-hMG vs. rFSH 1.85 [1.14;3.01] 0.013 Adjusting for progesterone at end-of- stimulationProgesterone (nmol/l) 0.84 [0.71; 0.99] 0.042 HP-hMG vs. rFSH 1.76[1.08; 2.87] 0.024 Adjusting for progesterone/estradiol atend-of-stimulation Progesterone/estradiol 0.26 [0.07; 0.95] 0.042 HP-hMGvs. rFSH 1.68 [1.02; 2.75] 0.040 Adjusting for number of follicles ≥12mm at end-of-stimulation Follicles ≥12 mm 1.05 [0.99; 1.10] 0.094 HP-hMGvs. rFSH 1.93 [1.18; 3.16] 0.008 Adjusting for endometrial thickness atend-of-stimulation Endometrial thickness (mm) 0.97 [0.85; 1.10] 0.653HP-hMG vs. rFSH 1.84 [1.13; 2.99] 0.014 Adjusting for number of oocytesretrieved Number of oocytes retrieved 1.00 [0.96; 1.04] 0.973 HP-hMG vs.rFSH 1.85 [1.13; 3.02] 0.014 Adjusting for top-quality embryo(s)/good-quality blastocyst(s) on transfer day ≥1 Top-quality embryo/good-qualityblastocyst 2.61 [1.48; 4.61] <0.001 HP-hMG vs. rFSH 1.80 [1.08; 3.00]0.025 OR = odds ratio; CI = Wald confidence interval ^(a)Two-sided Waldtest of OR = 1 (no effect).

These results show that the prevalence of patients with a high ovarianresponse (i.e. ≥15 oocytes retrieved) was approximately three timeshigher in women with high AMH (≥5.2 ng/ml) than in women in the non-highAMH category in both the long GnRH agonist and GnRH antagonist protocol.Although women with high AMH showed ovarian hyper-response in both theantagonist and long agonist protocols, ovarian stimulation with HP-hMGwas associated with a substantially lower high-response rate thanstimulation with rFSH. Hence, the risk of developing a high responsewhere an excessive response is predicted may be reduced by approximatelyone third by using HP-hMG instead of FSH for controlled ovarianstimulation, even in the GnRH antagonist protocol.

Without being bound by any theory, it is possible that the more moderateovarian response with HP-hMG, and the consequently reduced risk ofhyper-response with HP-hMG compared with rFSH, may be attributed atleast in part to the different FSH isoform profiles of HP-hMG and rFSH,which may influence the in vivo biopotency in humans and thereby therate of high ovarian response among the potential high-responders. Othercontributing factors may be the LH activity provided by HP-hMG that isnot present in rFSH, and the post-menopausal FSH isoform profile foundin the FSH activity of HP-hMG.

The logistic regression analyses in the overall population did notidentify any specific variable(s) that explained the different livebirth rates between HP-hMG and rFSH stimulation, but indicated thatprogesterone levels and progesterone/estradiol ratios at the end ofstimulation and the availability of top-quality embryos/good-qualityblastocysts (as assessed morphologically; aneuploidy was not assessed)influenced live birth rates.

Further analysis of the results suggested to the inventors that the useof HP-hMG for controlled ovarian stimulation has a direct effect onimproved oocyte quality and may reduce aneuploidy rate, and indicatethat progesterone, progesterone/estradiol ratio and embryo quality playa role in treatment outcome in patients at risk of hyper-response basedon high serum AMH levels.

Example 2—Prospective Clinical Trial

A randomized, assessor-blind phase IV clinical trial comparing HP-hMGand rFSH in a GnRH antagonist cycle with compulsory single-blastocysttransfer in a high responder subject population is planned for theUnited States. The aim of this study is to demonstrate the HP-hMG is atleast non-inferior to rFSH with respect to ongoing pregnancy rate (OPR)in potential high-responders undergoing IVF/ICSI treatment. Subjectswill be prospectively classified as potential high ovarian respondersbased on a serum level of AMH≥5.0 ng/mL by the Beckmann-Coulter Gen 2assay as described in Arce et al., Fertility and Sterility 99: 1644-53(2013), using a single reference laboratory (ReproSource, Inc., Woburn,Mass.) utilizing materials and reagents from the Beckman Coulter-DSLassay (Chaska, Minn.).

The phase IV, randomized, open-label, assessor-blind, parallel-group,multicenter study will be conducted at approximately 25-30 infertilitycenters in the United States. Approximately 600 females will beenrolled. Subjects are to be from age 21 up to age 36 years with regularovulatory menstrual cycles of 21 to 35 days, with a body mass index(BMI) between 18 and 32 kg/m², or 18 and 30 kg/m², unexplained or mildmale factor infertility for a period of 21 year, eligible for ICSIaccording to the investigator, and AMH≥5.0 ng/mL. Additional inclusioncriteria may be having an AFC≥10, ≥15, ≥18, or ≥20 in both ovariescombined. Key exclusion criteria include endometriosis stage I-IV,severe male factor infertility, polycystic ovary syndrome (PCOS), andprevious poor response to a COS cycle. Additional exclusion criteria maybe having an AFC<8 or <10 in both ovaries combined.

The basic protocol is outline in FIG. 2 . Optionally, combination oralcontraceptive pills (COCPs) started in the previous menstrual cycle willbe continued for a maximum of 21 days. Subjects will be randomized 1:1to undergo COS with either HP-hMG or rFSH in a GnRH antagonist cycle.After discontinuing the COCPs, gonadotropins (either HP-hMG or rFSH)will be started on the 3^(rd) day of withdrawal bleeding but no soonerthan the 5^(th) day after discontinuation of COCPs. The gonadotropindose will be initiated at 150 IU for the first 5 days. From StimulationDay 6 onward, based on follicular response assessed by TVUS, dosing canbe adjusted every day as needed by 75 IU per adjustment. However, themaximum gonadotropin dose will be 300 IU/day; gonadotropin dosing cancontinue for a maximum of 20 days. Coasting is prohibited.

When the lead follicle is >14 mm in diameter, the GnRH antagonist(ganirelix acetate) will be initiated at a daily dose of 0.25 mg andcontinued throughout the gonadotropin treatment period. A singleinjection of 250 μg hCG (choriogonadotropin alfa) will be administeredto induce final follicular maturation as soon as 3 follicles of ≥17 mmdiameter are observed on TVUS. If a subject has >25 follicles, she willreceive a GnRH agonist trigger and all resultant blastocysts will bevitrified after trophoectoderm biopsy, with no fresh embryo transfer todecrease risk of OHSS.

Oocyte retrieval will take place roughly 36 hours after hCGadministration. Oocytes will be inseminated using partner sperm by ICSI4±1 hours after retrieval. Oocyte, embryo and blastocyst quality will beassessed daily for 5 days following oocyte retrieval. On Day 5 followingICSI, a single blastocyst of the best quality available will betransferred; all remaining blastocysts will be frozen using thevitrification method.

Trophoectoderm (TE) biopsy for preimplantation genetic screening (PGS),optionally with next generation sequencing (NGS) technology, will bedone with laser on Day 5 or Day 6 of expanded blastocyst, but resultswill not be used to determine selection of a blastocyst for freshtransfer.

The day after oocyte retrieval, vaginal progesterone inserts (100 mgtwice a day—Endometrin®; Ferring) will be initiated for luteal phasesupport and will continue until the day of the β-hCG test (10 to 15 daysafter blastocyst/embryo transfer). Luteal support may be continued untilongoing pregnancy is confirmed.

Biochemical pregnancy will be confirmed by a positive β-hCGapproximately 2 weeks after blastocyst transfer. Clinical pregnancy willbe confirmed by TVUS indicating at least one intrauterine gestationalsac with fetal heart beat at 5 to 6 weeks gestation. Ongoing pregnancywill be confirmed by at least one intrauterine viable fetus at 10 to 11weeks gestation.

For subjects with no ongoing pregnancy in the fresh cycle, single frozenblastocyst transfers can be initiated within 1 year of each subject'sstart of treatment. PGS results will be used to select the euploidblastocysts for frozen transfer. Frozen-thawed embryo replacement cycledata will be collected, including blastocyst transfer information, β-hCGtest, clinical pregnancy, and ongoing pregnancy

Post-trial follow-up will include collection of delivery information(live birth and neonatal health), which will be collected for allsubjects with an ongoing pregnancy in the fresh cycle or the 1-yearpost-randomization frozen-thawed embryo replacement cycles. Live birthrate after the fresh cycle and cumulative live birth rate after freshand 1-year post-randomization frozen-thawed embryo replacement cycleswill be evaluated as part of the post-trial follow-up.

The post-trial endpoints include:

-   -   Cumulative live birth rate for fresh and frozen blastocyst        transfer (defined as the proportion of subjects with at least 1        viable live birth greater >21 weeks gestation);    -   Live birth rate for fresh blastocyst transfer (defined as the        proportion of subjects with at least 1 viable live birth        greater >21 weeks gestation);    -   Early pregnancy loss rate in frozen blastocyst transfer is        defined as 2 positive β-hCG tests but no ongoing pregnancy at        10-11 weeks gestation in the frozen cycle;    -   Late pregnancy loss rate (defined as a confirmed ongoing        pregnancy but no viable live birth greater >21 weeks gestation);        and    -   Positive β-hCG rate, clinical pregnancy rate, and ongoing        pregnancy rate for frozen blastocyst transfers.

The HP-hMG used will be Menopur, provided as a vial with powder (75 IUFSH activity and 75 IU LH activity) and 2 pre-filled syringes withsolvent (each containing 1.1 mL). After reconstitution, each vialdelivers 75 IU of FSH activity and 75 IU of LH activity.

The FSH used will be recombinant FSH (GONAL-F), provided aspen andcartridges filled with either 300, 450 or 900 IU FSH activity/1.08 mLsolution for injection.

The other drugs used will be:

-   -   Ganirelix Acetate Injection, manufactured by Merck, provided as        a pre-filled syringe (0.5 mL) delivering 0.25 mg ganirelix. A        daily dose of 0.25 mg will be continued throughout the        gonadotropin treatment period.    -   Ovidrel (choriogonadotropin alfa), manufactured by EMD Serono,        provided as a pre-filled syringe (0.5 mL) delivering 250 μg        choriogonadotropin alfa, to be administered as a single        injection as soon as 3 follicles of ≥17 mm diameter are observed        on TVUS.

The primary end point is ongoing pregnancy rate in the fresh transfercycle, defined as presence of at least one intrauterine pregnancy with aviable fetus with a detectable fetal heartbeat at 10-11 weeks gestation.Secondary endpoints include:

-   -   biochemical pregnancy rate (positive β-hCG)    -   clinical pregnancy rate (transvaginal ultrasonography showing at        least one intrauterine gestational sac with fetal heartbeat at        5-6 weeks gestation)    -   Early pregnancy loss (defined as 2 positive β-hCG tests but no        ongoing pregnancy at 10-11 weeks gestation in the fresh cycle.)    -   live birth rate    -   follicular development    -   endocrine profile (serum estradiol [E2], progesterone [P4], hCG,        LH)    -   oocytes retrieved, fertilization rate, and embryo quality    -   aneuploidy rate    -   endometrial assessment by TVUS

The present inventors believe that this study will show that stimulationof women at risk of a high ovarian response to controlled ovarianstimulation with HP-hMG improves embryo quality, increases theproportion of euploid blastocysts, decreases the proportion of aneuploidblastocysts, increases ongoing pregnancy rates and/or increases livebirth rates, as compared to comparable methods using recombinantfollicle stimulating hormone (rFSH) as the gonadotropin. For example,the present inventors believe that this study will show that stimulationof women at risk of a high ovarian response to controlled ovarianstimulation with HP-hMG results in fewer eggs but a higher proportion ofeuploid blastocysts as compared to comparable methods using rFSH as thegonadotropin.

What is claimed is:
 1. A method for reducing early pregnancy loss afterin vitro fertilization and transfer of a fresh or frozen blastocystafter controlled ovarian stimulation of a woman at risk of a highovarian response to controlled ovarian stimulation, comprising:selecting a woman at risk of a high ovarian response to controlledovarian stimulation determined to have a serum anti-Mülllerian hormone(AMH) level greater than or equal to 5.0±0.5 ng/ml, when measured usinga Beckmann-Coulter Gen 2 assay or a comparable AMH level measured by adifferent method; and then administering to the selected woman an amountof highly purified menotropin (HP-hMG) effective to stimulate follicledevelopment.
 2. The method of claim 1, wherein the woman has an antralfollicle count (AFC) of greater than or equal to 10 in both ovariescombined, prior to stimulation.
 3. The method of claim 1, wherein thewoman has an antral follicle count (AFC) of greater than or equal to 15in both ovaries combined, prior to stimulation.
 4. The method of claim1, wherein the amount of HP-hMG administered is from 100-300 IU per dayfor from about 1 to about 20 days.
 5. The method of claim 1, wherein theamount of HP-hMG administered is from 100-200 IU per day for from about1 to about 20 days.
 6. The method of claim 1, wherein the HP-hMG isadministered for at least 5 days.
 7. The method of claim 1, furthercomprising administering a gonadotropin hormone releasing hormone (GnRH)agonist.
 8. The method of claim 1, further comprising administering agonadotropin hormone releasing hormone (GnRH) antagonist.
 9. The methodof claim 1, further comprising administering an amount of humanchorionic gonadotropin (hCG) effective to trigger ovulation.
 10. Themethod of claim 9, further comprising harvesting oocytes from the woman.11. The method of claim 10, further comprising in vitro fertilization ofharvested oocytes.
 12. The method of claim 11, wherein the in vitrofertilization comprises intra-cytoplasmic sperm injection (ICSI). 13.The method of claim 11, further comprising assessing the chromosomalquality of blastocysts obtained from the woman after in vitrofertilization of oocytes harvested from the woman.
 14. The method ofclaim 13, further comprising blastocyst transfer of a blastocystdetermined to be a euploid blastocyst.
 15. The method of claim 14,wherein the blastocyst transfer is fresh transfer.
 16. The method ofclaim 13, further comprising freezing a blastocyst determined to be aeuploid blastocyst.
 17. The method of claim 1, wherein the administeringcomprises administering HP-hMG at a dose of 150 IU/day for at least afirst 5 days of treatment.
 18. A method for reducing early pregnancyloss after in vitro fertilization after controlled ovarian stimulationof a woman at risk of a high ovarian response to controlled ovarianstimulation, comprising: selecting a woman at risk of a high ovarianresponse to controlled ovarian stimulation determined to have a serumanti-Müllerian hormone (AMH) level greater than or equal to 5.0±0.5ng/ml, when measured using a Beckmann-Coulter Gen 2 assay or acomparable AMH level measured by a different method; and thenadministering to the selected woman an amount of highly purifiedmenotropin (HP-hMG) effective to stimulate follicle development.
 19. Themethod of claim 18, wherein the amount of HP-hMG administered is from100-300 IU per day for from about 1 to about 20 days.